Study protocol of the GLOW study: maximising treatment options for recurrent glioblastoma patients by whole genome sequencing-based diagnostics—a prospective multicenter cohort study

Background Glioblastoma (GBM), the most common glial primary brain tumour, is without exception lethal. Every year approximately 600 patients are diagnosed with this heterogeneous disease in The Netherlands. Despite neurosurgery, chemo -and radiation therapy, these tumours inevitably recur. Currently, there is no gold standard at time of recurrence and treatment options are limited. Unfortunately, the results of dedicated trials with new drugs have been very disappointing. The goal of the project is to obtain the evidence for changing standard of care (SOC) procedures to include whole genome sequencing (WGS) and consequently adapt care guidelines for this specific patient group with very poor prognosis by offering optimal and timely benefit from novel therapies, even in the absence of traditional registration trials for this small volume cancer indication. Methods The GLOW study is a prospective diagnostic cohort study executed through collaboration of the Hartwig Medical Foundation (Hartwig, a non-profit organisation) and twelve Dutch centers that perform neurosurgery and/or treat GBM patients. A total of 200 patients with a first recurrence of a glioblastoma will be included. Dual primary endpoint is the percentage of patients who receive targeted therapy based on the WGS report and overall survival. Secondary endpoints include WGS report success rate and number of targeted treatments available based on WGS reports and number of patients starting a treatment in presence of an actionable variant. At recurrence, study participants will undergo SOC neurosurgical resection. Tumour material will then, together with a blood sample, be sent to Hartwig where it will be analysed by WGS. A diagnostic report with therapy guidance, including potential matching off-label drugs and available clinical trials will then be sent back to the treating physician for discussing of the results in molecular tumour boards and targeted treatment decision making. Discussion The GLOW study aims to provide the scientific evidence for changing the SOC diagnostics for patients with a recurrent glioblastoma by investigating complete genome diagnostics to maximize treatment options for this patient group. Trial registration: ClinicalTrials.gov Identifier: NCT05186064. Supplementary Information The online version contains supplementary material available at 10.1186/s12920-022-01343-4.


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Tumor type specific evidence TREATMENT LEVEL ±

KEYNOTE-158
High tumor mutation load Potential eligibility for DRUP is dependent on tumor type details therefore certain tumor types may not be eligible for the DRUP.
The iClusion knowledgebase is used to annotate DNA aberrations for potential clinical study eligibility. Please note clinical study eligibility depends on multiple patient and tumor characteristics of which only the DNA aberrations are considered in this report.
The Clinical Knowledgebase (CKB) is used to annotate variants of all types with clinical evidence. Only treatment associated evidence with evidence levels ( ( A FDA approved therapy and/or guidelines; B late clinical trials; C early clinical trials) can be reported. Potential evidence items with evidence level ( D case reports and preclinical evidence) are not reported.
The symbol ( ▲ ) means that the evidence is responsive. The symbol ( ▼ ) means that the evidence is resistant. The abbreviation ( P mentioned after the level of evidence) indicates the evidence is predicted responsive/resistent. More details about CKB can be found in their Glossary Of Terms

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Evidence on other tumor types PTEN partial loss 1, 2, 3, 4, 5, 6 The Clinical Knowledgebase (CKB) is used to annotate variants of all types with clinical evidence. Only treatment associated evidence with evidence levels ( ( A FDA approved therapy and/or guidelines; B late clinical trials; C early clinical trials) can be reported. Potential evidence items with evidence level ( D case reports and preclinical evidence) are not reported.
The symbol ( ▲ ) means that the evidence is responsive. The symbol ( ▼ ) means that the evidence is resistant. The abbreviation ( P mentioned after the level of evidence) indicates the evidence is predicted responsive/resistent. More details about CKB can be found in their Glossary Of Terms

Proficient 0
The HR-deficiency score is determined by CHORD, a WGS signature-based classifier comparing the signature of this sample with signatures found across samples with known BRCA1/BRCA2 inactivation. Tumors with a score greater or equal than 0.5 are considered HR deficient by complete BRCA inactivation.
Low High

Stable 0.12
The microsatellite stability score represents the number of somatic inserts and deletes in (short) repeat sections across the whole genome of the tumor per Mb. This metric can be considered as a good marker for instability in microsatellite repeat regions. Tumors with a score greater than 4.0 are considered microsatellite unstable (MSI).

High 189
The tumor mutational load represents the total number of somatic missense variants across the whole genome of the tumor. Patients with a mutational load over 140 could be eligible for immunotherapy within the DRUP study.

Molecular tissue of origin prediction
The title shows the conclusion of the prediction of the molecular tissue of origin. If none of the similarity predictions has a likelihood ≥80%, no reliable conclusion can be drawn ('results inconclusive').
The left plot shows the likelihoods (similarity) for all the origin types analyzed by the molecular tissue of origin prediction tool. Only when the likelihood is ≥80% (a peak in the green outer band of the plot), a reliable prediction (with >95% accuracy) can be drawn. Lower likelihoods (<80%) suggest there is similarity with that tissue of origin, but this is less strong and there is lower confidence.
The right plot(s) shows the breakdown of the strongest predicted likelihood(s) into the contribution of the 1) SNV types (related to those used in Cosmic signatures), 2) driver landscape and passenger characteristics (e.g. tumor-type specific drivers), and 3) somatic mutation pattern (mutation distribution across the genome).

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The outer first circle shows the chromosomes. The third circle shows all observed tumor purity adjusted copy number changes, including both focal and chromosomal events. Copy number losses are indicated in red, green shows regions of copy number gain. The scale ranges from 0 (complete loss) to 6 (high level gains). If the absolute copy number is > 6 it is shown as 6 with a green dot on the diagram.
The fourth circle represents the observed 'minor allele copy numbers' across the chromosome. The range of the chart is from 0 to 3. The expected normal minor allele copy number is 1, and anything below 1 is shown as a loss and represents a LOH event (orange). Minor allele copy numbers above 1 indicate amplification events of both A and B alleles at the indicated locations (blue).
The innermost circle displays the observed structural variants within or between the chromosomes. Translocations are indicated in blue, deletions in red, insertions in yellow, tandem duplications in green and inversions in black.

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Details on the report in general Details on the reported clinical evidence

Details on reported somatic variants
The analysis is based on reference genome version GRCh37.
Transcripts used for reporting can be found on https://resources.hartwigmedicalfoundation.nl in directory 'Patient-Reporting' and are generally the canonical transcripts as defined by Ensembl.
Variant detection in samples with lower tumor content is less sensitive. In case of a low tumor purity (below 20%) likelihood of failing to detect potential variants increases.

The (implied) tumor purity is the percentage of tumor cells in the tumor material based on analysis of whole genome data.
The Clinical Knowledgebase (CKB) (https://ckbhome.jax.org/) is used to annotate variants of all types with clinical evidence, with a hyperlink to the specific evidence items when available. The evidence is gathered from CKB without further checks or interpretation. This also means that if a certain evidence item or drugbiomarker is missing from the knowledgebase it will also not be included in this report. More details about CKB can be found in their Glossary Of Terms. Hartwig Medical Foundation is not responsible for the content of the knowledgebases used to generate this report. Furthermore, Hartwig Medical Foundation is not liable and cannot be held accountable for any incorrectness, incompleteness or error of any other kind in the knowledgebases, or the external software used to harmonize and curate the knowledgebases.
The 'Read Depth' displays the raw number of reads supporting the variant versus the total number of reads on the mutated position.
The 'Copies' field indicates the number of alleles present in the tumor on this particular mutated position.
The 'tVAF' field displays the variant allele frequency corrected for tumor purity.
The 'Biallelic' field indicates whether the variant is present across all alleles in the tumor (and is including variants with loss-of-heterozygosity).
The 'Driver' field is based on the driver probability calculated based on the HMF database. A variant in a gene with High driver likelihood is likely to be positively selected for during the oncogenic process.

Details on reported gene fusions Details on reported gene disruptions
The lowest copy number value along the exonic regions of the canonical transcript is determined as a measure for the gene's copy number.
Copy numbers are corrected for the implied tumor purity and represent the number of copies in the tumor DNA.
Any gene with less than 0.5 copies along the entire canonical transcript is reported as a full loss.
Any gene where only a part along the canonical transcript has less than 0.5 copies is reported as a partial loss.
Any gene with more copies than 3 times the average tumor ploidy along the entire canonical transcript is reporte as a full gain.
Any gene where only a part of the canonical transcript has more copies than 3 times the average tumor ploidy is reported as a partial gain.
The canonical, or otherwise longest transcript validly fused is reported.
Fusions are restricted to those in the HMF known fusion list and can be found on https://resources.hartwigmedicalfoundation.nl in directory 'Patient-Reporting'.
We additionally select fusions where one partner is promiscuous in either 5' or 3' position.
The 'Driver' field is set to HIGH in case the fusion is a known pathogenic fusion, or otherwise a fusion where the promiscuous partner is fused in an exon range that is typically observed in literature. All other fusions get assigned a LOW driver likelihood.
Genes are reported as being disrupted if their canonical transcript has been disrupted.
The range of the disruption is indicated by the intron/exon/promoter region of the break point and the direction the disruption faces.

The type of disruption can be INV (inversion), DEL (deletion), DUP (duplication), INS (insertion), SGL (single) or BND (translocation).
A gene for which no wild type exists anymore in the tumor DNA due to disruption(s) is reported in a separate section called 'homozygous disruptions'.

Details on reported pharmacogenetics
Virusses will be reported if they are present in our reporting database as clinically relevant (HPV, MCV, HBV, EBV and HHV-8) and DNA integration for the virus can be detected. If the virus is clinically relevant and no DNA integration is found, the following conditions must be met: -Percentage covered of the viral genome is >90% -Coverage of the virus is higher than the expected clonal mean coverage Reporting of EBV is independent of tumor integration. This means that to be reportable, the viral EBV genome must be covered >90% and the coverage of the virus must be higher than the expected clonal mean coverage.
See the directory 'Patient Reporting' in https://resources.hartwigmedicalfoundation.nl for details on the panel and for more links to advice on treatment adjustments.
The called haplotypes for a gene are the simplest combination of haplotypes that perfectly explains all of the observed variants for that gene. If no combination of haplotypes in the panel can perfectly explain the observed variants, then 'Unresolved Haplotype' is called.
Wild type is assumed when no variants are observed.

Sample details
The samples have been sequenced at Hartwig Medical Foundation, Science Park 408, 1098XH Amsterdam The samples have been analyzed by Next Generation Sequencing using Whole Genome Sequencing The HMF sample ID is: PNT00012345T The germline reporting choice of this patient is: no reporting The results in this report have been obtained between 01-Oct-2020 and 10-Dec-2021 This experiment is performed on the tumor sample which arrived on 05-Oct-2020 with internal tumor barcode FR12345678 This experiment is performed on the blood sample which arrived on 01-Oct-2020 with internal blood barcode FR12123488 The results stated in this report are based on the tested tumor and blood sample.
This experiment is performed according to lab procedures: PREP013V23-QC037V20-SEQ008V25 This report was generated by Lieke Schoenmaker (trained IT employee) and checked by a trained Clinical Molecular Biologist in Pathology (KMBP) This report is addressed to: PI, HMF Testing Center, 1000 AB AMSTERDAM Comments: This is a test report and is based on COLO829. Where is referred to CKB, VICC evidence is listed due to licensing restrictions.

Disclaimer
The data on which this report is based is generated from tests that are performed under ISO/ICE-17025:2017 TESTING L633 accreditation and have passed all internal quality controls.
This report is generated by patient reporter version 7.24 based on HMF-FOR-080.
The OncoAct user manual can be found at https://www.oncoact.nl/manual.
This report is based on pipeline version 5.25.
The 'primary tumor location' and 'primary tumor type' have influence on the clinical evidence/study matching. No check is performed to verify the received information.
The conclusion of this report is based solely on the results of the DNA sequencing of the tumor and the received tumor type. Final interpretation of the clinical consequence of this report should therefore always be performed by the treating physician.
Based on a tumor purity of at least 20%, the test has a sensitivity of >95% for detection of somatic variants and >95% for detection of translocations and gene copy number changes.
For feedback or complaints please contact qualitysystem@hartwigmedicalfoundation.nl.
For questions about the contents of this report, please contact diagnosticssupport@hartwigmedicalfoundation.nl.

Edwin Cuppen,
Director Hartwig Medical Foundation